SUMMARY/ABSTRACT (30 lines of text) The goal of this research is to identify the mechanistic origins of a newly-discovered type of hematopoietic (blood- forming) stem cell (HSC), which we have named the latent-HSC. HSCs are defined by functional assays, the gold-standard being transplantation into an irradiated (bone marrow-compromised) recipient. HSCs have two key functional characteristics: (1) multipotency, readout from the regeneration of the lympho-myeloid blood system in the recipient; (2) self-renewal, readout by stable blood system reconstitution, with long-term HSC activity confirmed by serial transplantation. HSCs are thought to be responsible for life-long blood system homeostasis, through gradual differentiation into cell-amplifying lineage-restricted progenitors. However, aging is associated with blood system deterioration, most notably loss of lymphoid immune cell production and myeloid malignancies, with age-related HSC dysfunction proposed as a key underlying mechanism. In a recent clonal analysis of the aged mouse HSC compartment, we identified a functionally-novel type of HSC. We have termed this population ?latent-HSCs? because following primary transplantation, latent-HSCs display only myeloid-restricted output, but following serial transplantation display full multipotent lympho-myeloid output. My laboratory was only able to detect latent-HSCs because we have pioneered HSC analysis at five-blood lineage resolution. Traditionally, HSCs have only been defined by neutrophil/monocyte, T and B cell output, but ignored the two most abundant blood cell types, platelets and erythrocytes. Latent-HSCs often only display platelet/erythrocyte output in primary recipients, undetectable by traditional methods. Surprisingly, latent-HSCs are more frequent than long-term multipotent HSCs in the aged bone marrow, but have not been detected in young mice. Such a cell type that gains multipotency with age (and serial transplantation) is difficult to reconcile with current models of hematopoiesis. However, our findings suggest there may be a reservoir of latent lymphoid potential in the aged bone marrow. By developing approaches to activate latent-HSCs, we may identify therapeutic strategies to boost the immune system in the elderly and extend healthy aging. This exploratory R21 research project aims to identify the origin of latent-HSCs, to better understand the biological and translational relevance of this novel age-specific stem cell population. We will test three hypotheses: (1) that the latent-HSC phenotype could be a consequence of transplanting aged myeloid-restricted stem/progenitors into a ?rejuvenating? young recipient bone marrow; (2) that latent-HSCs could be generated from myeloid stem/progenitors by somatic mutations that lead to acquisition of lymphoid potential; and (3) that primary transplantation stress could be insufficient to induce multipotent differentiation of aged latent-HSCs. This new research avenue into the cellular mechanisms of HSC aging aims to identify strategies to ?rejuvenate? stem cell activity and prevent age-related loss of immune function. In summary, the study will set a new direction in stem cell aging research, and will lead to new models and assays in biomedical science.